The visualization of cellular gene expression in normal and diseased tissues, combined with histological and pathological analysis, is a critical step towards understanding biological gene function and its contribution to disease processes. Immunohistochemistry (IHC), which uses antibodies specific to differentially expressed gene products (or biomarkers) best represents this discipline. Instead of analyzing proteins, mRNA transcripts can be detected by in situ hybridization (ISH), which in principle offers more flexibility in (1) probe synthesis (either as RNA transcript or synthetic oligonucleotide), (2) control of specificity by probing different segments of the same transcript, and (3) detection of non-coding RNAs and analysis of mRNA isoforms. However, conventional RNA ISH protocols are difficult to standardize because tissue sample preparation methods and storage affect RNA integrity and its detection due to loss of fragmented RNA by diffusion from tissue sections during the process of ISH. We have re-analyzed the critical components of the ISH process including RNA stability, RNA fixation, probe design and hybridization, signal amplification and detection, and established a research program to correct its weaknesses. Our developments already resulted in a robust protocol to retain and detect miRNAs in tissue sections by crosslinking the miRNA 5'-monophosphate to protein amino acid side chains via phosphoramidate bond formation. Here, we demonstrate that ISH of mRNA and its fragments has to be addressed by similar approaches to increase retention in tissue sections. Recent advances in such fields as genome and RNA sequencing, oligonucleotide probe design, automation of tissue processing, and fluorescence microscopy scanning and image processing, now position RNA ISH as a technology to rapidly surpass conventional IHC. Providing reliable reagents and demonstrations of proof of concepts will have a profound impact on the development of molecular diagnostics and biomarkers in cancer research. Other medical areas with a need for tissue-based diagnostics, such as fibrosis of various organs linked to chronic viral infection or transplantation would also benefit from reliable RNA ISH. As a proof of principle, we will apply the approach to improve sarcoma diagnosis through more accurate classification of small blue round cell tumors (SBRCTs) and spindle cell tumors (SCTs) utilizing RNA FISH for monitoring transcripts of diagnostic and prognostic value emerging from the sarcoma genome project. These studies will be carried out in close collaboration with the Sarcoma Disease Management Team and the Soft Tissue Sarcoma PO1 Investigators at MSKCC led by Samuel Singer. In summary, we are building the chemical foundation for a sustainable discovery and diagnostic RNA FISH platform that will rival conventional IHC due to its flexibility in RNA probe production and multiplexing at reduced cost and time. It will unite classical histology and advanced molecular pathology, thereby increasing the value of archival tissue collections with linked clinical records. PUBLIC HEALTH RELEVANCE: This project describes new approaches to better measure gene expression at the RNA level in normal and diseased tissues and unites classical histology and advanced molecular pathology. Collaborative studies will be conducted with the Sarcoma Disease Management Team at Memorial Sloan Kettering Cancer Center with the aim to facilitate sarcoma diagnosis through more accurate classification of gene expression in different types of small blue round cell tumors and spindle cell tumors.